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The Journal of Nutrition
Community and International Nutrition
Zinc Deficiency Is Common among
Healthy Women of Reproductive Age in
Bhaktapur, Nepal1,2
Ram K. Chandyo,3,4 Tor A. Strand,4,6* Maria Mathisen,4 Manjeswori Ulak,3 Ramesh K. Adhikari,3
Bjørn J. Bolann,5,7 and Halvor Sommerfelt4,8
3
Department of Child Health, Institute of Medicine, Tribhuvan University, 2533 Kathmandu, Nepal; 4Centre for International Health,
and 5Institute of Medicine, University of Bergen, 5020 Bergen, Norway; 6Medical Microbiology, Department of Laboratory
Medicine, Sykehuset Innlandet, 2609 Lillehammer, Norway; 7Laboratory of Clinical Biochemistry, Haukeland University Hospital,
5021 Bergen, Norway; and 8Division of Infectious Disease Control, Norwegian Institute of Public Health, 0403 Oslo, Norway
Zinc deficiency is a major public health problem in many developing countries. However, its prevalence is still unknown in
most populations. Women of reproductive age in developing countries are highly vulnerable to nutritional deficiencies,
including that of zinc. To estimate the prevalence of zinc deficiency and to identify important dietary sources of zinc, we
undertook a cross-sectional survey in 500 nonpregnant Nepalese women and measured their plasma zinc concentrations.
We also examined the associations between plasma zinc and dietary intake of zinc or phytate, iron status, plasma
concentrations of C-reactive protein, albumin, and hemoglobin. Food intake was estimated by 2 24-h dietary recalls and
1 FFQ for each woman. The plasma zinc concentration was (mean 6 SD) 8.5 6 2.4 mmol/L and more than three-quarters of
the women were zinc deficient. Dietary zinc intake did not predict plasma zinc concentration, whereas phytate intake was
negatively and significantly associated with plasma zinc. The other variables that were associated with plasma zinc were
plasma albumin and hemoglobin concentration. Rice contributed 50% to the total estimated daily zinc intake and wheat
and meat each contributed 15%. Rice also contributed 68% to the daily intake of phytate. In conclusion, we found that zinc
deficiency was common in women of reproductive age and that the foods contributing substantial amounts of zinc also
contributed importantly to the intake of phytate. J. Nutr. 139: 594–597, 2009.
Introduction
Zinc is an essential trace element with a key role in numerous
basic cellular functions in humans. It is crucial to the normal
function of the immune system (1,2) and is involved in DNA
synthesis, cellular division, proliferation, and growth (3). Zinc
is also required during pregnancy for optimal growth and
development of the fetus and for maternal tissue expansion
(4). Poor maternal zinc status has been associated with
negative pregnancy outcomes (5–7), including spontaneous
abortion, congenital malformation, low birth weight, and
preterm delivery (8–10). Micronutrient deficiencies in early
pregnancy, including that of zinc, are common among Nepali
women (11).
Traditionally, the Nepali diet is monotonous and cereal
based and consists of limited amounts of food from animal
sources. Cereal-based diets are high in phytate, which inhibits
zinc absorption and the inhibitory effect is particularly high
1
Supported by Norwegian Universities Committee for Development, Research
and Education grant number 36/2002 and the Research Council of Norway grant
numbers 160854 and 172226.
2
Author disclosures: R. Adhikari, B. Bolann, R. Chandyo, M. Mathisen,
H. Sommerfelt, T. A. Strand, and M. Ulak, no conflicts of interest.
* To whom correspondence should be addressed. E-mail: [email protected]
594
when the phytate:zinc (P:Z)9 molar ratio in the diet is .15
(12).
Data on zinc deficiency based on population surveys are still
lacking from many developing countries (13,14). Less precise
estimates, such as those based on national food balance sheets
and on the prevalence of clinical manifestations of zinc deficiency,
like stunting and diarrhea in children, have been used instead
(15). However, these proxies are influenced by several factors and
are rather unspecific markers of zinc deficiency and probably not
suitable for studying an adult population.
Our objective in this study was to assess the prevalence
of zinc deficiency by measuring plasma zinc concentrations in
a random sample of 500 women of childbearing age living in
Bhaktapur, Nepal. We also measured plasma albumin and
C-reactive protein (CRP) concentrations, because most of the
intravascular zinc is bound to albumin and the concentration of
plasma zinc is influenced by inflammation (16). In a subsample,
we also administered 2 24-h dietary recalls to identify important
sources of zinc and phytate.
9
Abbreviations used: CRP, C-reactive protein; CF, carpet factory; GAM,
generalized additive model; ICP-AES, inductively coupled plasma atomic emission spectrometry; P:Z, phytate:zinc.
0022-3166/08 $8.00 ª 2009 American Society for Nutrition.
Manuscript received November 10, 2008. Initial review completed November 21, 2008. Revision accepted December 21, 2008.
First published online January 21, 2009; doi:10.3945/jn.108.102111.
Downloaded from jn.nutrition.org at Univ I Bergen/Norway on March 3, 2009
Abstract
Subjects and Methods
The study was approved by the ethical board of Institute of Medicine,
Tribhuvan University in Katmandu, Nepal and the Human Research Ethics
Committee of the Medical Faculty at the University of Bergen, Norway.
Study area and food habits. From September 2000 to November
2001, we recruited women from the Bhaktapur municipality in the
Kathmandu valley, Nepal. This is a semiurban, agricultural-based town
with 80% of the population constituted by the Newar ethnic group.
Around the town of Bhaktapur, there are ;50 carpet factories (CF) in
which migrant families from different ethnic groups, mainly Tamang and
Magar, live and work for longer or shorter periods. The CF workers have
become an important part of the population in Bhaktapur and were
therefore also included in our study.
Sample size. We expected a prevalence of zinc deficiency of .25%. A
sample size of 450 women is required to detect this prevalence with a
lower 95% confidence limit of 21%. We assumed that we would be unable
to obtain an adequate blood specimens from ;10% of the women and
therefore targeted a sample size of 500.
Laboratory analysis. Blood was collected from the cubital vein
between 0900 and 1500 (72% of the specimens before noon) in
micronutrient-free heparinized polypropylene tubes (Sarstedt). Within
10 min of collection, the heparinized blood was centrifuged (760 3 g;
10 min, room temperature), separated, and the plasma transferred to
micronutrient-free polypropylene vials (Eppendorf). These vials were
initially refrigerated at the field clinic for a maximum of 5 h, transported
on ice to the university hospital the same day, and stored at 245C until
they were transferred on dry ice to Norway.
After thawing, the plasma specimens were analyzed for zinc using
inductively coupled plasma atomic emission spectrometry (ICP-AES)
from Thermo Jarell-Ash at the Laboratory for Clinical Biochemistry,
Haukeland Hospital, Bergen, Norway. Spectrascan Certified Element
Standard for Atomic Spectroscopy (Teknolab) was used as the reference
standard. All specimens were analyzed twice and the mean concentration
was used. The CV between the analyses was ,6.5%. Plasma CRP and
albumin levels were measured using an immunoturbidimetric (TinaQuant, Roche) and a Bromcresol Green colorimetric assay, respectively,
on a Modular P analyzer (Roche Diagnostics). Two vials contained too
little material to obtain reliable zinc concentrations.
Statistical analysis. The data were double entered into Microsoft
VisualFoxPro databases with computerized logic, range, and consistency
checks. The associations between plasma zinc and the variables of interest were described by the Spearman correlation coefficients. P , 0.05
was considered significant.
Descriptive statistics and linear regression analyses were undertaken
using Stata, version 9 (STATA Corp) and, when appropriate, adjusted for
the design effects induced by stratification and clustering. The figure
describing the relationship between plasma zinc and plasma albumin
concentration was constructed using generalized additive models (GAM)
in the statistical software R, version 1.9.0. We also undertook crude and
multiple GAM analyses to assess whether any of the associations were
linear or confounded by other variables (22). Values in the text are
means 6 SD unless otherwise noted.
Results
The plasma zinc concentration did not differ between local
resident and CF women and this stratification variable did
not modify any of the associations described in this article. The
pooled data are therefore presented.
Subject characteristics. A total of 296 (59%) women were
married and, among these, 209 (71%) used contraceptives,
mainly Depo-Provera. The weight of the participants was 48.8 6
7.5 kg and their height was 149.6 6 5.8 cm; 86 (17%) of the
women were shorter than 145 cm. Thirty-five (7%) of the women
were fasting (no meals, snack, or tea before sampling) and onethird reported that they did not have a morning meal prior to
blood sample collection. Among the women who reported having
a morning meal (67%), the duration between the last meal and
blood collection was 2.3 h (range, 0.55–6.25 h). Similarly, 60%
of women reported having had tea, whereas 22% had a snack
before blood sampling (Table 1).
TABLE 1
General characteristics of the nonpregnant
women included in a study on zinc status in
Bhaktapur, Nepal1
Characteristics
Age, y
Married, n (%)
Hemoglobin, g/L
Hemoglobin ,120 g/L, n (%)
Plasma ferritin ,15 mg/L, n (%)
Plasma CRP, mg/L
Land owner, n (%)
Schooling, y
BMI, kg/m2
Smoker,2 n (%)
Illiterate, n (%)
Daily wage earners, n (%)
Vegetarians,2 n (%)
23 6 6
296 (59)
132 6 13
58 (12)
98 (20)
0 (0, 0.8)
333 (67)
4 (0, 8)
21.8 6 3.0
26 (7)
166 (33)
247 (49)
10 (3)
1
Definitions. Zinc deficiency was defined as a plasma zinc concentration
,11.3 mmol/L for samples obtained in the morning from fasting women,
Values are means 6 SD, n ¼ 500 unless otherwise indicated, n (%), or median
(interquartile range).
2
Information based on 379 women from whom we obtained dietary recalls.
Zinc status among women in Bhaktapur, Nepal
595
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Selection procedures and dietary recalls. The details of the selection
procedures and dietary recall methods are provided elsewhere (17).
Because of frequent migration, we were concerned that CF women
would be underrepresented. To ensure that this important and marginalized group was adequately represented, we used separate sampling
frames for the CF women and for the local residents.
The study included nonpregnant women aged 13–35 y, without any
ongoing disease, who were living in the Bhaktapur municipality. Pregnancy
status was assessed by asking about the date of the last menstruation and by
a urine test for pregnancy, whenever necessary. We excluded women with
acute (e.g. fever, diarrhea, dysentery) or chronic (e.g. tuberculosis, diabetes,
hypertension) illness and those taking vitamins, minerals, or drugs (with the
exception of hormonal contraceptives). From the lists of women in the 2
strata, we randomly selected and approached 792 women. We enrolled 500
of these women, 403 of whom were from the stratum consisting of local
residents and 97 from the CF stratum. In 379 of the 500 enrolled women, we
also administered a FFQ and 2 24-h dietary recalls ;1 wk apart and on
different weekdays. The daily intakes of the various nutrients from the 24-h
dietary recalls were calculated using Indian food tables from the Wfood2
program version 1.0 (18). Ideally, all the study subjects should be fasting
before blood was collected, but this was not possible. We recorded the time
of the last meal or snack before the women visited the clinic. The women
were weighed using a UNICEF electronic scale (SECA) with an accuracy of
100 g and the height was measured using a locally made wooden board that
measured height to the nearest cm.
,10.7 mmol/L for samples obtained in the morning from women who
did not fast, and ,9.3 mmol/L for samples obtained in the afternoon
(15). The estimated average requirement of zinc using an unrefined plant
based diet is 9 mg/d for women aged 13–18 y and 7 mg/d for
nonpregnant women older than 18 y (19). Women consuming less that
these cutoffs were considered to have inadequate intake. The P:Z molar
ratio in the diet reflects the inhibitory effect of phytate on zinc absorption
and was estimated using a standard algorithm (20,21).
TABLE 2
Plasma concentrations of zinc and albumin and
intakes of zinc and phytate in nonpregnant women
in Bhaktapur, Nepal1
Variables
Values
Plasma zinc concentration, mmol/L
,11.3 mmol/L in morning fasting samples, %
,10.7 mmol/L in morning nonfasting samples, %
,9.3 mmol/L in afternoon samples, %
Plasma albumin, g/L
,35 g/L, %
Intake of nutrients, n ¼ 379
Zinc, mg/d
,7 mg/d, % (95% CI ) (women .18 y )
,9 mg/d, % (95% CI ) (women 13–18 y )
Phytate, mg/d
P:Z molar ratio
1
8.5 6 2.4
88 (29 of 33)
90 (291 of 324)
78 (110 of 141)
42.5 6 3.1
1 (0.2)
8.6 6 3.3
29 (23, 34)
69 (58, 79)
2198 6 695
26.4 6 5.9
Values are means 6 SD, n ¼ 500 unless otherwise indicated.
Plasma zinc and its relation with plasma albumin and intake of zinc and phytate. The plasma zinc concentration was
8.5 6 2.4 mmol/L and the 2.5th and 97.5th percentiles were 5.3
and 14.1 mmol/L, respectively. Overall, depending on fasting status,
78–90% of the specimens had plasma zinc concentrations that
indicated zinc deficiency (Table 2). We depict the association
between plasma zinc and plasma albumin concentration (Fig. 1).
Zinc intake was not associated with plasma zinc concentration in our study. The only nutrient intake that was associated
with plasma zinc concentration was phytate, which was negatively correlated (r ¼ 20.15; P ¼ 0.003). However, the P:Z molar
ratio was not associated with the plasma zinc concentration.
TABLE 3
Zinc
Rice grain, flake, or flour
Wheat grain or flour (refined and unrefined)
Meat (buffalo, chicken, goat)
Green or dry vegetables (mustard, radish, spinach, etc.)
Pulses and beans (lentil, black and red gram)
Potatoes
Milk products (buffalo or cow)
Eggs
2
3
Values are means, ranges, n, or percentages.
Total 758 dietary recalls (2 recalls from each woman).
Data based on FFQ from 394 women.
596
Discussion
The results of our study indicate that more than three-quarters of
the apparently healthy, nonpregnant women were zinc deficient
as defined by low plasma zinc concentration. Similar prevalences
of zinc deficiency have been reported among nonpregnant (23)
and pregnant (11,24) Indian and Nepalese women.
The prevalence of iron deficiency anemia has been suggested
as a proxy for zinc deficiency, because meat and other animal
flesh foods are rich sources of both minerals (25). However, in
this population, the prevalence of anemia and iron deficiency
(17) was substantially lower than the prevalence of zinc deficiency. This was also found in a recent study in Ethiopian
women (26).
We measured plasma zinc levels using ICP-AES. Although a
study by Dipietro et al. (27) demonstrated a satisfactory agreement between ICP-AES and atomic absorption spectrometry,
which is more frequently used for determining plasma zinc
concentrations, we cannot rule out the possibility that ICP-AES
and atomic absorption spectrometry give somewhat different
readouts and, accordingly, different prevalence estimates.
The P:Z molar ratio was very high in our study, indicating a
potential for profound inhibition of intestinal zinc absorption.
Most of the intake of phytate was from rice (68%) and the
phytate content was estimated from uncooked rice. Phytate is to
some extent lost during cooking and we might accordingly have
overestimated the phytate content in the food. However, a study
from India suggests that cooking induces only a limited lowering
of phytate concentration (28). The intake of zinc in our study
Main sources of zinc and phytate in nonpregnant women in Bhaktapur, Nepal1
Foods
1
Multiple regression analyses. The crude associations presented here were not substantially altered when the independent
variables were included in multiple regression models. The results
from the multiple regression models are therefore not presented.
Chandyo et al.
Phytate
mg/100 g
1.1
353–786
2.0–2.3
620–845
3.2
0
0.1–0.8
20–42
0.8–1.3
255–358
0.3
81
0.3
0
1.2
0
Recalls with
particular foods2
Women who reported
consuming food item
at least once a week3
Zinc
contribution
n (%)
758 (100)
314 (41)
142 (19)
517 (68)
294 (39)
569 (75)
500 (65)
69 (9)
Phytate
contribution
%
394
255
159
319
256
347
259
130
(100)
(65)
(40)
(81)
(65)
(88)
(66)
(33)
50
15
15
6
3
3
3
1
68
18
0
5
3
4
0
0
Downloaded from jn.nutrition.org at Univ I Bergen/Norway on March 3, 2009
Nutrient intake and P:Z molar ratio. Seventy-six percent
(95% CI: 72%, 81%) of the women had energy intakes less than
the recommended dietary allowance of 9205 kJ. The intake of
zinc and phytate and the P:Z molar ratio are presented (Table 2).
The interquartile range of zinc intake was 7.2–9.4 mg. A total of
29% of women .18 y of age and 69% of women #18 y of age
had an inadequate intake of zinc. We present the main sources of
zinc and phytate and their intake frequency (Table 3). Rice
contributed 50% to the total daily zinc intake and wheat and
meat each contributed 15%. Rice also contributed 68% to the
intake of phytate. The P:Z molar ratio in our study was 26.4 6
5.9. The P:Z molar ratio was .15 in .90% of the women.
Plasma zinc status and its relation with iron status and
intake of iron. Intakes of iron and zinc were strongly correlated
(r ¼ 0.79; P , 0.001). The Spearman rank correlation
coefficients between zinc intake and plasma ferritin, plasma
transferrin receptor, and hemoglobin concentration were 0.17
(P ¼ ,0.001), 20.10 (P ¼ 0.056), and 0.26 (P , 0.001),
respectively. The plasma zinc concentration was associated with
plasma hemoglobin (r ¼ 0.16; P , 0.001) but not with plasma
transferrin receptor (r ¼ 20.01; P ¼ 0.9) or ferritin concentrations (r ¼ 0.07; P ¼ 0.08).
4.
5.
6.
7.
8.
9.
10.
11.
13.
14.
15.
was not associated with plasma zinc concentration. Plasma zinc
reflects an individual’s usual zinc intake over a few weeks or
months (29). Meat, which in this population was the food item
that had the highest concentration of bioavailable zinc, was
consumed by most women but not on a regular basis. This
relatively high intra-person variability in consumption of zincdense foods in combination with the very high level of phytate
intake could result in a weaker association between zinc intake
or the P:Z molar ratio with plasma zinc. Furthermore, we have
used Indian food tables from Wfood2 to calculate the intake of
zinc (18). The zinc and phytate content of the local foods are probably somewhat different, but we think that this is the best available tool in the absence of Nepalese food composition tables.
In conclusion, our study indicates that there is a high prevalence of zinc deficiency in women of reproductive age in
Bhaktapur, Nepal. This may increase the risk of infections and
poor pregnancy outcomes in these women. Moreover, food that
contributed most to the intake of zinc also contributed substantially to the intake of phytate, which seemed to have a negative
impact on their zinc status.
Acknowledgments
We thank Shyam S. Dhaubhadel, founder chairman of Siddhi
Memorial Hospital in Bhaktapur, for his cooperation in undertaking the study and Irene Ro Iversen at the Laboratory for
Clinical Biochemistry at Haukeland University Hospital for
proficient processing of the plasma specimens.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
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